Skip to main content
. 2016 Sep 29;291(46):24041–24053. doi: 10.1074/jbc.M116.744722

FIGURE 4.

FIGURE 4.

Alternative cleavage of human APP harboring FAD mutations by β-secretase, and the resultant generation of Aβ. A, levels of APP and APP CTFs in cells. N2a cells expressing hAPP (WT), Swedish hAPP K670N/M671L, hAPP A673V, or Icelandic hAPP A673T were lysed, and cell lysates (10 μg of protein) were analyzed by Western blotting with anti-APP antibody and anti-α-tubulin antibody (to confirm equal protein loading). Mature (N- and O-glycosylated form) and immature (N-glycosylated form) hAPP (upper), CTFβ/C99, CTFβ′/C89, and CTFα/C83 (middle), and α-tubulin (lower) are indicated. Numbers indicate molecular size markers (in kDa). The levels of APP and APP CTFβ/C99 are indicated as ratios relative to the levels of hAPP (WT), which was assigned a reference value 1.0. Statistical analysis was performed using Dunnett's multiple comparison test. p values are provided for comparison with the hAPP wild-type (WT) (mean ± S.E., n = 6; *, p < 0.05). B, levels of Aβ(1–40) and Aβ(1–42) secreted from cells. Aβ levels in culture media of cells expressing hAPP variants, as indicated in panel A, were assayed by sELISA. Statistical analysis was performed using Dunnett's multiple comparison test. p values are provided for the comparison with the hAPP wild-type (WT) (mean ± S.E., n = 6; **, p < 0.01; ***, p < 0.001). C, representative MS spectra of Aβ with amino-terminal β- and β′-cleavage sites generated from hAPP with or without FAD mutation. Aβ(1-XX) is the product of cleavage at the β-site, whereas Aβ(11-XX) is the product of cleavage at the β′-site. D, relative levels of Aβ(11–40) production and the Aβ(11–40)/Aβ(1–40) ratio. Levels of Aβ(11–40) production from each APP are indicated as ratios relative to the level of hAPP (WT), which was assigned a reference value of 1.0 (left). The Aβ(11–40)/Aβ(1–40) ratio is also indicated as a relative ratio (right). Statistical significance was determined by Dunnett's multiple comparisons test (n = 6), and p values are indicated (**, p < 0.01; ***, p < 0.001). Data are shown as mean ± S.E.